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Abstract:

1,3-Dipole-functional compounds (e.g., azide functional compounds) can be
reacted with certain alkynes in a cyclization reaction to form
heterocyclic compounds. Useful alkynes (e.g., strained, cyclic alkynes)
and methods of making such alkynes are also disclosed. The reaction of
1,3-dipole-functional compounds with alkynes can be used for a wide
variety of applications including the immobilization of biomolecules on a
substrate.

Claims:

1. A compound of the formula: ##STR00014## wherein: each R1 is
independently selected from the group consisting of hydrogen, halogen,
hydroxy, alkoxy, nitrate, nitrite, sulfate, and a C1-C10 organic group;
each R2 is independently selected from the group consisting of
hydrogen, halogen, hydroxy, alkoxy, nitrate, nitrite, sulfate, and a
C1-C10 organic group; X represents C═O, C═N--OR3,
C═N--NR3R4, CHOR3, or CHNHR3; each R3 and
R4 independently represents hydrogen or an organic group; and
R8 represents an organic group.

2. The compound of claim 1 wherein R8 represents a biomolecule.

3. The compound of claim 2 wherein the biomolecule is selected from the
group consisting of peptides, proteins, glycoproteins, nucleic acids,
lipids, saccharides, oligosaccharides, polysaccharides, and combinations
thereof.

7. The compound of claim 6 wherein the organic dye is a fluorescent dye.

8. A compound of the formula: ##STR00015## wherein: each R1 is
independently selected from the group consisting of hydrogen, halogen,
hydroxy, alkoxy, nitrate, nitrite, sulfate, and a C1-C10 organic group;
each R2 is independently selected from the group consisting of
hydrogen, halogen, hydroxy, alkoxy, nitrate, nitrite, sulfate, and a
C1-C10 organic group; X represents CHOR3; R3 represents
--C(O)Z, wherein: Z represents an alkyl group, OR6, or NHR7;
R6 is selected from the group consisting of an alkyl group, an aryl
group, an alkaryl group, and an aralkyl group; R7 is a biotinylation
product of a primary amine-containing organic group; and R8
represents an organic group.

9. The compound of claim 8 wherein the biotinylation product is the
biotinylation product of a primary amine-containing group of the formula
--(CH2CH2O)b(CH2)c-Ld-(CH2CH2O).s-
ub.e(CH2)fNH2 and/or
--(CD2CD2O)b(CD2)c-Ld-(CD2CD2O).s-
ub.c,(CD2)fNH2, wherein b=0 to 100; c=0 to 100; d=0 to 100;
e=0 to 100; f=0 to 100; and L is an optional cleavable linker.

10. The compound of claim 9 wherein the cleavable linker, if present, is
a disulfide.

11. A compound of the formula: ##STR00016## wherein: each R1 is
independently selected from the group consisting of hydrogen, halogen,
hydroxy, alkoxy, nitrate, nitrite, sulfate, and a C1-C10 organic group;
each R2 is independently selected from the group consisting of
hydrogen, halogen, hydroxy, alkoxy, nitrate, nitrite, sulfate, and a
C1-C10 organic group; X represents C═O, C═N--OR3,
C═N--NR3R4, CHOR3, or CHNHR3; R3 represents
a polymeric or a copolymeric group; R4 independently represents
hydrogen or an organic group; and R8 represents an organic group.

12. The compound of claim 11 wherein the copolymeric group comprises a
hydrophilic segment and a hydrophobic segment.

13. The compound of claim 11 wherein the copolymeric group comprises a
fragment of the formula
--[CH2CH2O]n--[C(O)(CH2)5O]m--H, wherein
n=0 to 100 and m=0 to 100.

14. A dibenzocyclooctyne having a biotin fragment attached thereto.

15. The dibenzocyclooctyne of claim 14 having the formula: ##STR00017##
wherein: each Rl and R2 is hydrogen; X represents CHOR3;
and R3 represents an organic group comprising a biotin fragment.

16. A compound of the formula: ##STR00018## wherein: each R1 and
R2 is hydrogen; X represents CHOR3; R3 represents an
organic group comprising a biotin fragment; and R8 represents an
organic group.

18. The dibenzocyclooctyne of claim 17 having the formula: ##STR00019##
wherein: each R1 and R2 is hydrogen; X represents CHOR3;
and R3 represents an organic group comprising a covalently bound
organic dye.

19. A compound of the formula: ##STR00020## wherein: each R1 and
R2 is hydrogen; X represents CHOR3; R3 represents an
organic group comprising a covalently bound organic dye; and R8
represents an organic group.

20. A dibenzocyclooctyne having a nanoparticle attached thereto.

21. The dibenzocyclooctyne of claim 20 having the formula: ##STR00021##
wherein: each R1 is independently selected from the group consisting
of hydrogen, halogen, hydroxy, alkoxy, nitrate, nitrite, sulfate, and a
C1-C10 organic group; each R2 is independently selected from the
group consisting of hydrogen, halogen, hydroxy, alkoxy, nitrate, nitrite,
sulfate, and a C1-C10 organic group; X represents C═N--OR3,
C═N--NR3R4, CHOR3, or CHNHR3; R3 represents
an organic group attached to a nanoparticle; and R4 represents
hydrogen or an organic group.

22. A method of preparing a heterocyclic compound, the method comprising:
combining at least one azide-functional compound with at least one
dibenzocyclooctyne according to claim 20; and allowing the at least one
azide-functional compound and the at least one alkyne to react under
conditions effective to form the heterocyclic compound.

23. A compound of the formula: ##STR00022## wherein: each R1 is
independently selected from the group consisting of hydrogen, halogen,
hydroxy, alkoxy, nitrate, nitrite, sulfate, and a C1-C10 organic group;
each R2 is independently selected from the group consisting of
hydrogen, halogen, hydroxy, alkoxy, nitrate, nitrite, sulfate, and a
C1-C10 organic group; X represents C≡N--OR3,
C═N--NR3R4, CHOR3, or CHNHR3; R3 represents
an organic group attached to a nanoparticle; R4 represents hydrogen
or an organic group; and R8 represents an organic group.

24. An alkyne of the formula: ##STR00023## wherein: each R1 and
R2 is hydrogen; X represents CHOR3; and R3 represents an
organic group.

25. A compound of the formula: ##STR00024## wherein: each R1 and
R2 is hydrogen; X represents CHOR3; R3 represents an
organic group; and R8 represents an organic group.

[0003] Bioorthogonal reactions are reactions of materials with each other,
wherein each material has limited or substantially no reactivity with
functional groups found in vivo. The efficient reaction between an azide
and a terminal alkyne, i.e., the most widely studied example of "click"
chemistry, is known as a useful example of a bioorthogonal reaction. In
particular, the Cu(I) catalyzed 1,3-dipolar cyclization of azides with
terminal alkynes to give stable triazoles (e.g., Binder et al., Macromol.
Rapid Commun. 2008, 29:952-981) has been employed for tagging a variety
of biomolecules including proteins, nucleic acids, lipids, and
saccharides. The cycloaddition has also been used for activity-based
protein profiling, monitoring of enzyme activity, and the chemical
synthesis of microarrays and small molecule libraries.

[0004] An attractive approach for installing azides into biomolecules is
based on metabolic labeling whereby an azide containing biosynthetic
precursor is incorporated into biomolecules using the cells' biosynthetic
machinery. This approach has been employed for tagging proteins, glycans,
and lipids of living systems with a variety of reactive probes. These
probes can facilitate the mapping of saccharide-selective glycoproteins
and identify glycosylation sites. Alkyne probes have also been used for
cell surface imaging of azide-modified bio-molecules and a particularly
attractive approach involves the generation of a fluorescent probe from a
non-fluorescent precursor by a [3+2] cycloaddition.

[0005] Despite the apparent utility of reacting an azide with a terminal
alkyne, applications in biological systems using this reaction have been
practically limited by factors including the undesirable presence of a
copper catalyst. Thus, there is a continuing, unmet need for new
bioorthogonal reactions.

SUMMARY

[0006] In one aspect, the present invention provides an alkyne, and
methods of making an alkyne. In one embodiment, the alkyne is of the
formula:

##STR00001##

wherein: each R1 is independently selected from the group consisting
of hydrogen, halogen, hydroxy, alkoxy, nitrate, nitrite, sulfate, and a
C1-C10 organic group; each R2 is independently selected from the
group consisting of hydrogen, halogen, hydroxy, alkoxy, nitrate, nitrite,
sulfate, and a C1-C10 organic group; X represents C═O,
C═N--OR3, C═N--NR3R4, CHOR3, or CHNHR3;
and each R3 and R4 independently represents hydrogen or an
organic group (e.g., which can include a cleavable linker). Also provided
are blends of certain alkynes with a polymer or a copolymer that can
optionally form a copolymer micelle, which can be useful, for example,
for controlling the delivery of drugs as described herein.

[0007] In another embodiment, the alkyne includes: a cleavable linker
fragment including at least two ends; an alkyne fragment attached to a
first end of the cleavable linker fragment; and a biotinylated fragment
attached to a second end of the cleavable linker fragment. In preferred
embodiments, the alkyne fragment includes a strained, cyclic alkyne
fragment. In certain embodiments, the alkyne further includes at least
one heavy mass isotope. Optionally, the alkyne further includes at least
one detectable label such as a fluorescent label.

[0008] Alkynes such as those described herein above can be reacted with at
least one 1,3-dipole-functional compound (e.g., an azide-functional
compound, a nitrile oxide-functional compound, a nitrone-functional
compound, an azoxy-functional compound, and/or an acyl diazo-functional
compound) in a cyclization reaction to form a heterocyclic compound,
preferably in the substantial absence of added catalyst (e.g., Cu(I)).
Optionally, the reaction can take place within or on the surface of a
living cell. In certain embodiments, the at least one
1,3-dipole-functional compound includes a 1,3-dipole-functional
biomolecule such as a peptide, protein, glycoprotein, nucleic acid,
lipid, saccharide, oligosaccharide, and/or polysaccharide. Optionally,
the 1,3-dipole-functional biomolecule includes a detectable label such as
an affinity label. The heterocyclic compounds formed by the alkyne with
the at least one 1,3-dipole-functional compound are also disclosed
herein. In certain embodiments, the reaction between the alkyne and the
at least one 1,3-dipole-functional compound can take place within or on
the surface of a living cell.

[0009] For embodiments in which the heterocyclic compound includes a
biotinylated fragment, the heterocyclic compound can be bound to a
compound that binds biotin, such as avidin and/or streptavidin.

[0010] In another aspect, the present invention provides a substrate
having an alkyne as described herein on the surface thereof. The
substrate can be in the form of a resin, a gel, nanoparticles, or
combinations thereof. Optionally, the substrate is a three-dimensional
matrix. In preferred embodiments, the X group of an alkyne of Formula I
represents a point of attachment to the surface of the substrate. Such
substrates can be useful for immobilizing biomolecules such as peptides,
proteins, glycoproteins, nucleic acids, lipids, saccharides,
oligosaccharides, and/or polysaccharides. Articles including an
immobilized biomolecule, such as a protein immobilized on a
three-dimensional matrix, are also disclosed herein.

[0011] The compositions and methods disclosed herein can offer advantages
over bioorthogonal reactions known in the art. See, for example, Baskin
et al., QSAR Comb. Sci. 2007, 26:1211-1219. For example, alkynes of
Formula I as described herein (e.g., wherein X represents C═O,
C≡N--OR3, C═N--NR3R4, CHOR3, or
CHNHR3; and each R3 and R4 independently represents
hydrogen or an organic group) surprisingly have been found to have higher
reactivity towards 1,3-dipole-functional compounds than other strained,
cyclic alkynes (e.g., wherein X represents CH2). See, for example,
Codelli, et al., J. Am. Chem. Soc. 2008, 130:11486-11493; Johnson et al.,
Chem. Commun. 2008, 3064-3066; Sletten et al., Organic Letters 2008,
10:3097-3099; and Laughlin et al., Science 2008, 320:664-667. Further,
convenient methods having the flexibility to prepare a wide variety of
alkynes of Formula I are disclosed herein. In addition, alkynes of
Formula I have the capability of reacting not only with azides, but also
a variety of other 1,3-dipole-functional compounds.

Definitions:

[0012] The term "comprises" and variations thereof do not have a limiting
meaning where these terms appear in the description and claims.

[0013] As used herein, "a," "an," "the," "at least one," and "one or more"
are used interchangeably.

[0014] As used herein, the term "or" is generally employed in the sense as
including "and/or" unless the context of the usage clearly indicates
otherwise.

[0015] Also herein, the recitations of numerical ranges by endpoints
include all numbers subsumed within that range (e.g., 1 to 5 includes 1,
1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).

[0016] The above summary is not intended to describe each disclosed
embodiment or every implementation of the present invention. The
description that follows more particularly exemplifies illustrative
embodiments. In several places throughout the application, guidance is
provided through lists of examples, which examples can be used in various
combinations. In each instance, the recited list serves only as a
representative group and should not be interpreted as an exclusive list.

[0022] FIG. 6 illustrates embodiments of cell-surface labeling with
compounds 2 and 9. Jurkat cells grown for three days in the absence or
presence of Ac4ManNAz (25 micromolar) were incubated a) with
compounds 2 and 9 (30 micromolar) for 0-180 minutes or b) with compounds
2 and 9 (0-100 micromolar) for 1 hour at room temperature. Next, the
cells were incubated with avidin-FITC for 15 minutes at 4° C.,
after which cell lysates were assessed for fluorescence intensity.
Samples are indicated as follows: blank cells incubated with 2
(∘) or 9 (quadrature), and Ac4ManNAz cells incubated
with 2 ( ) or 9 (.box-solid.).

[0023]FIG. 7 illustrates an embodiment of toxicity assessment of cell
labeling procedure and cycloaddition reaction with compound 9. Jurkat
cells grown for 3 days in the absence (a) or presence (b) of
Ac4ManNAz (25 micromolar) were incubated with compound 9 (0-100
micromolar) for 1 hour at room temperature. The cells were washed three
times and then incubated with avidin conjugated with fluorescein for 15
minutes at 4° C., after which cells were washed three times. Cell
viability was assessed at different points during the procedure with
trypan blue exclusion; after incubation with 9 (black), after avidin-FITC
incubation (grey), and after complete procedure (white). Treatment with
Cu1Cl (1 mM) under the same conditions led to approximately 98% cell
death for both the blank and the Ac4ManNAz treated cells.

[0038]FIG. 22 illustrates cycloadditions of 4-dibenzocyclooctynol with
various nitrones. Compounds were mixed at 1:1 molar ratio at a final
concentration of 6mM and reacted for a time indicated.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0039] Alkynes such as those described herein can be reacted with at least
one 1,3-dipole-functional compound in a cyclization reaction to form a
heterocyclic compound. In preferred embodiments, the reaction can be
carried out in the substantial absence of added catalyst (e.g., Cu(I)).
Exemplary 1,3-dipole-functional compounds include, but are not limited
to, azide-functional compounds, nitrile oxide-functional compounds,
nitrone-functional compounds, azoxy-functional compounds, and/or acyl
diazo-functional compounds.

[0040] Exemplary alkynes include alkynes of the formula:

##STR00002##

wherein: each R1 is independently selected from the group consisting
of hydrogen, halogen, hydroxy, alkoxy, nitrate, nitrite, sulfate, and a
C1-C10 organic group (and preferably a C1-C10 organic moiety); each
R2 is independently selected from the group consisting of hydrogen,
halogen, hydroxy, alkoxy, nitrate, nitrite, sulfate, and a C1-C10 organic
group (and preferably a C1-C10 organic moiety); X represents C═O,
C═N--OR3, C═N--NR3R4, CHOR3, or CHNHR3;
and each R3 and R4 independently represents hydrogen or an
organic group (and in some embodiments an organic moiety). In preferred
embodiments, each R1 represents hydrogen and/or each R2
represents hydrogen. Optionally, R3 includes a covalently bound
organic dye (e.g., a fluorescent dye).

[0041] As used herein, the term "organic group" is used for the purpose of
this invention to mean a hydrocarbon group that is classified as an
aliphatic group, cyclic group, or combination of aliphatic and cyclic
groups (e.g., alkaryl and aralkyl groups). In the context of the present
invention, suitable organic groups for compounds of this invention are
those that do not interfere with the reaction of an alkyne with a
1,3-dipole-functional compound to form a heterocyclic compound. In the
context of the present invention, the term "aliphatic group" means a
saturated or unsaturated linear or branched hydrocarbon group. This term
is used to encompass alkyl, alkenyl, and alkynyl groups, for example. The
term "alkyl group" means a saturated linear or branched monovalent
hydrocarbon group including, for example, methyl, ethyl, n-propyl,
isopropyl, tart-butyl, amyl, heptyl, and the like. The term "alkenyl
group" means an unsaturated, linear or branched monovalent hydrocarbon
group with one or more olefinically unsaturated groups (i.e.,
carbon-carbon double bonds), such as a vinyl group. The term "alkynyl
group" means an unsaturated, linear or branched monovalent hydrocarbon
group with one or more carbon-carbon triple bonds. The teen "cyclic
group" means a closed ring hydrocarbon group that is classified as an
alicyclic group, aromatic group, or heterocyclic group. The term
"alicyclic group" means a cyclic hydrocarbon group having properties
resembling those of aliphatic groups. The term "aromatic group" or "aryl
group" means a mono- or polynuclear aromatic hydrocarbon group. The term
"heterocyclic group" means a closed ring hydrocarbon in which one or more
of the atoms in the ring is an element other than carbon (e.g., nitrogen,
oxygen, sulfur, etc.).

[0042] As a means of simplifying the discussion and the recitation of
certain terminology used throughout this application, the tennis "group"
and "moiety" are used to differentiate between chemical species that
allow for substitution or that may be substituted and those that do not
so allow for substitution or may not be so substituted. Thus, when the
term "group" is used to describe a chemical substituent, the described
chemical material includes the unsubstituted group and that group with
nonperoxidic O, N, S, Si, or F atoms, for example, in the chain as well
as carbonyl groups or other conventional substituents. Where the term
"moiety" is used to describe a chemical compound or substituent, only an
unsubstituted chemical material is intended to be included. For example,
the phrase "alkyl group" is intended to include not only pure open chain
saturated hydrocarbon alkyl substituents, such as methyl, ethyl, propyl,
tert-butyl, and the like, but also alkyl substituents bearing further
substituents known in the art, such as hydroxy, alkoxy, alkylsulfonyl,
halogen atoms, cyano, nitro, amino, carboxyl, etc. Thus, "alkyl group"
includes ether groups, haloalkyls, nitroalkyls, carboxyalkyls,
hydroxyalkyls, sulfoalkyls, etc. On the other hand, the phrase "alkyl
moiety" is limited to the inclusion of only pure open chain saturated
hydrocarbon alkyl substituents, such as methyl, ethyl, propyl,
tert-butyl, and the like.

[0043] Alkynes of Formula I are typically strained, cyclic alkynes.
Surprisingly it has been found that alkynes of Formula I as described
herein (e.g., wherein X represents C═O, C═N--OR3,
C═N--NR3R4, CHOR3, or CHNHR3; and each R3
and R4 independently represents hydrogen or an organic group) have
been found to have higher reactivity towards 1,3-dipole-functional
compounds than other strained, cyclic alkynes (e.g., wherein X represents
CH2).

[0044] In certain embodiments of alkynes of Formula I, X can represent
C═N--OR3 wherein R3 is an organic group. For example,
R3 can have the formula --(CH2)aC(O)Y, wherein: a is 1-3;
Y represents OH or NHR5; and R5 represents hydrogen or a
biotinylation product of a primary amine-containing organic group. The
primary amine-containing group can, for example, be of the formula
--(CH2CH2O)b(CH2)c-Ld-(CH2CH2O).s-
ub.e(CH2)fNH2 and/or
--(CD2CD2O)b(CD2)c-Ld-(CD2CD2O).s-
ub.e(CD2)fNH2, wherein b=0 to 100 (e.g., 10 to 100); c=0 to
100 (and preferably 1 to 10); d=0 to 100 (and preferably 1 to 10); e=0 to
100 (e.g., 10 to 100); f=0 to 100 (and preferably 1 to 10); and L is an
optional cleavable linker (e.g., a disulfide).

[0045] In certain embodiments of alkynes of Formula I, X can represent
CHOR3, wherein R3 is selected from the group consisting of an
alkyl group, an aryl group, an alkaryl group, and an aralkyl group. For
example, R3 can have the formula --C(O)Z, wherein: Z represents an
alkyl group, OR6, or NHR7; and R6 and R7 are each
independently selected from the group consisting of an alkyl group, an
aryl group, an alkaryl group, and an aralkyl group. In certain
embodiments, R7 can be a biotinylation product of a primary
amine-containing organic group. The primary amine-containing group can,
for example, be of the formula
--(CH2CH2O)b(CH2)c-Ld-(CH2CH2O).s-
ub.e(CH2)fNH2 and/or
--(CD2CD2O)b(CD2)-Ld-(CD2CD2O)e(C-
D2)fNH2, wherein b=0 to 100 (e.g., 10 to 100); c=0 to 100
(and preferably 1 to 10); d=0 to 100 (and preferably 1 to 10); e=0 to 100
(e.g., 10 to 100); f=0 to 100 (and preferably 1 to 10); and L is an
optional cleavable linker (e.g., a disulfide).

[0046] An exemplary alkyne of Formula I is the species in which X
represents C═O, an alkyne of the formula:

##STR00003##

[0047] Another exemplary alkyne of Formula I is the species in which X
represents CHOH, an alkyne of the formula:

##STR00004##

[0048] Another exemplary alkyne of Formula I is the species in which X
represents CHNH2, an alkyne of the formula:

##STR00005##

[0049] Another exemplary alkyne of Formula I is the species in which X
represents C═N--OR3, an alkyne of the formula:

##STR00006##

wherein R3 represents hydrogen or an organic group (and in some
embodiments an organic moiety).

[0050] Additional exemplary alkynes include alkynes that have: a cleavable
linker fragment including at least two ends; an alkyne fragment attached
to a first end of the cleavable linker fragment; and a biotinylated
fragment attached to a second end of the cleavable linker fragment. In
certain embodiments, the alkyne fragment includes a strained, cyclic
alkyne fragment. In certain embodiments, the alkyne further includes at
least one heavy mass isotope. Optionally, the alkyne further includes at
least one detectable label (e.g., a fluorescent label).

[0051] In certain embodiments of alkynes of Formula I, X can represent a
polymeric or a copolymeric group. For embodiments in which X represents a
copolymeric group, the copolymeric group can include a hydrophilic
segment and a hydrophobic segment. For example, the copolymeric group can
include a fragment of the formula
--[CH2CH2O]n--[C(O)(CH2)m--H, wherein n=0 to 100
(e.g., 10 to 100) and m=0 to 100 (e.g., 10 to 100). Surfaces on which
drops of water or aqueous solutions exhibit a contact angle of less than
90 degrees are commonly referred to as "hydrophilic." The contact angle
of a hydrophobic material with water is typically greater than 90
degrees.

[0052] Exemplary methods of making alkynes of Formula I are also disclosed
herein. In one embodiment, the method includes: brominating an alkene of
the formula:

##STR00007##

to provide a dibromide of the formula:

##STR00008##

and dehydrobrominating the dibromide of Formula XV to provide the alkyne
of the formula:

##STR00009##

wherein: each R1 is independently selected from the group consisting
of hydrogen, halogen, hydroxy, alkoxy, nitrate, nitrite, sulfate, and a
C1-C 10 organic group; each R2 is independently selected from the
group consisting of hydrogen, halogen, hydroxy, alkoxy, nitrate, nitrite,
sulfate, and a C1-C10 organic group; X represents C═O,
C═N--OR3, C═N--NR3R4, CHOR3, or CHNHR3;
and each R3 and R4 independently represents hydrogen or an
organic group (e.g., which can include a cleavable linker).

[0053] A wide variety of 1,3-dipole-functional compounds can be used to
react with the alkynes disclosed herein. As used herein, a
"1,3-dipole-functional compound" is meant to include compounds having at
least one 1,3-dipole group attached thereto. As used herein, a
"1,3-dipole group" is intended to refer to a group having a three-atom
pi-electron system containing 4 electrons delocalized over the three
atoms. Exemplary 1,3-dipole groups include, but are not limited to,
azides, nitrile oxides, nitrones, azoxy groups, and acyl diazo groups. In
certain embodiments, the 1,3-dipole-functional compound can be a
biomolecule having at least one 1,3-dipole group attached thereto.
Optionally, the at least one 1,3-dipole-functional compound can include a
detectable label (e.g., an immunoassay or affinity label).

[0054] One or more 1,3-dipole-functional compounds (e.g., azide-functional
compounds, nitrile oxide-functional compounds, nitrone-functional
compounds, azoxy-functional compounds, and/or acyl diazo-functional
compounds) can be combined with an alkyne as described herein under
conditions effective to react in a cyclization reaction and form a
heterocyclic compound. Preferably, conditions effective to form the
heterocyclic compound can include the substantial absence of added
catalyst. Conditions effective to form the heterocyclic compound can also
include the presence or absence of a wide variety of solvents including,
but not limited to, aqueous (e.g., water) and non-aqueous solvents;
protic and aprotic solvents; polar and non-polar solvents; and
combinations thereof. The heterocyclic compound can be formed over a wide
temperature range, with a temperature range of 0° C. to 40°
C. (and in some embodiments 23° C. to 37° C.) being
particularly useful when biomolecules are involved. Conveniently,
reaction times can be less than one day, and sometimes one hour or even
less.

[0055] In certain embodiments, the cyclization reaction between the one or
more 1,3-dipole-functional compounds and the alkyne can take place within
or on the surface of a living cell. Such reactions can take place in vivo
or ex vivo. As used herein, the term "in vivo" refers to a reaction that
is within the body of a subject. As used herein, the term "ex vivo"
refers to a reaction in tissue (e.g., cells) that has been removed, for
example, isolated, from the body of a subject. Tissue that can be removed
includes, for example, primary cells (e.g., cells that have recently been
removed from a subject and are capable of limited growth or maintenance
in tissue culture medium), cultured cells (e.g., cells that are capable
of extended growth or maintenance in tissue culture medium), and
combinations thereof.

[0056] An exemplary embodiment of a 1,3-dipole-functional compound is an
azide-functional compound of the formula R8--N3 (e.g.,
represented by the valence structure R8---N--N═N+),
wherein R8 represents and organic group (e.g., a biomolecule).
Optionally, R8 can include a detectable label (e.g., an affinity
label).

[0057] The cyclization reaction of an azide-functional compound of the
formula R8--N3 with an exemplary alkyne of Formula I can result
in one or more heterocyclic compounds of the formulas:

##STR00010##

wherein: each R1 is independently selected from the group consisting
of hydrogen, halogen, hydroxy, alkoxy, nitrate, nitrite, sulfate, and a
C1-C10 organic group; each R2 is independently selected from the
group consisting of hydrogen, halogen, hydroxy, alkoxy, nitrate, nitrite,
sulfate, and a C1-C10 organic group; X represents C═O,
C═N--OR3, C═N--NR3R4, CHOR3, or CHNHR3;
each R3 and R4 independently represents hydrogen or an organic
group (e.g., which can include a cleavable linker); and R8
represents an organic group (e.g., which can include a biomolecule and
optionally a cleavable linker).

[0058] Another exemplary embodiment of a 1,3-dipole-functional compound is
a nitrile oxide-functional compound of the formula R8--CNO (e.g.,
represented by the valence structure R8--+C═N--O-),
wherein R8 represents and organic group (e.g., a biomolecule).
Optionally, R8 can include a detectable label (e.g., an affinity
label).

[0059] The cyclization reaction of a nitrile oxide-functional compound of
the formula R8--CNO with an exemplary alkyne of Formula I can result
in one or more heterocyclic compounds of the formulas:

##STR00011##

wherein: each R1 is independently selected from the group consisting
of hydrogen, halogen, hydroxy, alkoxy, nitrate, nitrite, sulfate, and a
C1-C1 0 organic group; each R2 is independently selected from the
group consisting of hydrogen, halogen, hydroxy, alkoxy, nitrate, nitrite,
sulfate, and a C1-C10 organic group; X represents C═O,
C═N--OR3, C═N--NR3R4, CHOR3, or CHNHR3;
each R3 and R4 independently represents hydrogen or an organic
group (e.g., which can include a cleavable linker); and R8
represents an organic group (e.g., which can include a biomolecule and
optionally a cleavable linker).

[0060] Another exemplary embodiment of a 1,3-dipole-functional compound is
a nitrone-functional compound of the formula
(R10)2CN(R10)O (e.g., represented by the valence structure
(R10)2C═+N(R10)--O-), wherein each R10
independently represents hydrogen or an organic group, with the proviso
that at least one R10 represents an organic group (e.g., a
biomolecule). Optionally, at least one R10 can include a detectable
label (e.g., an affinity label).

[0061] The cyclization reaction of a nitrone-functional compound of the
formula) (R10)2CN(R10)O with an exemplary alkyne of
Formula I can result in one or more heterocyclic compounds of the
formulas:

##STR00012##

wherein: each R1 is independently selected from the group consisting
of hydrogen, halogen, hydroxy, alkoxy, nitrate, nitrite, sulfate, and a
C1-C10 organic group; each R2 is independently selected from the
group consisting of hydrogen, halogen, hydroxy, alkoxy, nitrate, nitrite,
sulfate, and a C1-C10 organic group; X represents C═O,
C═N--OR3, C═N--NR3R4, CHOR3, or CHNHR3;
and' each R3, R4, and R10 independently represents
hydrogen or an organic group, with the proviso that at least one R10
represents an organic group (e.g., which can include a biomolecule and
optionally a cleavable linker).

[0062] Another exemplary embodiment of a 1,3-dipole-functional compound is
an azoxy-functional compound of the formula R10--NN(R10)O
(e.g., represented by the valence structure
R10--N═+N(R10)--O-), wherein each R10
independently represents hydrogen or an organic group, with the proviso
that at least one R10 represents an organic group (e.g., a
biomolecule). Optionally, at least one R10 can include a detectable
label (e.g., an affinity label).

[0063] The cyclization reaction of an azoxy-functional compound of the
formula R10--NN(R10)O with an exemplary alkyne of Formula I can
result in one or more heterocyclic compounds of the formulas:

##STR00013##

wherein: each R1 is independently selected from the group consisting
of hydrogen, halogen, hydroxy, alkoxy, nitrate, nitrite, sulfate, and a
C1-C10 organic group; each R2 is independently selected from the
group consisting of hydrogen, halogen, hydroxy, alkoxy, nitrate, nitrite,
sulfate, and a C1-C10 organic group; X represents C═O,
C═N--OR3, C═N--NR3R4, CHOR3, or CHNHR3;
and each R3, R4, and R10 independently represents hydrogen
or an organic group, with the proviso that at least one R10
represents an organic group (e.g., which can include a biomolecule and
optionally a cleavable linker).

[0064] For embodiments in which the heterocyclic compound formed from the
cyclization reaction between the alkyne and the one or more
1,3-dipole-functional compounds includes a detectable label, the
heterocyclic compound can be detected using the detectable label. For
example, for embodiments in which the detectable label is an affinity
label, affinity binding (e.g., affinity chromatography) can be used to
detect the heterocyclic compound.

[0065] In addition, for embodiments in which the heterocyclic compound
formed from the cyclization reaction between the alkyne and the one or
more 1,3-dipole-functional compounds includes a biotinylated fragment,
the heterocyclic compound can be bound by contacting the heterocyclic
compound with a compound that binds biotin (e.g., avidin and/or
streptavidin). Further, the bound heterocyclic compound can be detected
by methods described herein.

[0066] Cyclization reactions between alkynes as disclosed herein and
1,3-dipole-functional compounds can be used for a wide variety of
applications. For example, an alkyne as disclosed herein can be attached
to the surface of a substrate. In certain embodiments, the X group of the
alkyne represents a point of attachment to the surface of the substrate.
One of skill in the art will recognize that the X group can
advantageously be selected to include functionality (e.g., biotin,
activated esters, activated carbonates, and the like) to enable
attachment of the alkyne to a functional substrate (e.g., amine
functionality, thiol functionality, and the like) through a wide variety
of reactions.

[0067] Substrates having an alkyne attached to the surface thereof can be
reacted with 1,3-dipole-functional compounds to form heterocyclic
compounds, effectively chemically bonding the 1,3-dipole-functional
compounds to the substrate. Such substrates can be, for example, in the
form of resins, gels, nanoparticles (e.g., including magnetic
nanoparticles), or combinations thereof. In certain embodiments, such
substrates can be in the form of microarrays or even three-dimensional
matrices or scaffolds. Exemplary three-dimensional matrices include, but
are not limited to, those available under the trade designations
ALGIMATRIX 3D Culture system, GELTRIX matrix, and GIBCO three-dimensional
scaffolds, all available from Invitrogen (Carlsbad, Calif.). Such
three-dimensional matrices can be particularly useful for applications
including cell cultures.

[0068] 1,3-Dipole-functional biomolecules (e.g., 1,3-dipole-functional
peptides, proteins, glycoproteins, nucleic acids, lipids, saccharides,
oligosaccharidcs, and/or polysaccharides) can be immobilized on, and
preferably covalently attached to, a substrate surface by contacting the
1,3-dipole-functional biomolecules with a substrate having an alkyne
attached to the surface thereof under conditions effective for a
cyclization reaction to form a heterocyclic compound. Preferably,
conditions effective to form the heterocyclic compound can include the
substantial absence of added catalyst. Conditions effective to form the
heterocyclic compound can also include the presence or absence of a wide
variety of solvents including, but not limited to, aqueous (e.g., water
and other biological fluids) and non-aqueous solvents; protic and aprotic
solvents; polar and non-polar solvents; and combinations thereof. The
heterocyclic compound can he formed over a wide temperature range, with a
temperature range of 0° C. to 40° C. (and in some
embodiments 23° C. to 37° C.) being particularly useful.
Conveniently, reaction times can be less than one day, and sometimes one
hour or even less.

[0069] For example, when the substrate is in the form of a
three-dimensional matrix and the 1,3-dipole-functional biomolecule is a
1,3-dipole-functional protein (e.g., an azide-functional protein), the
cyclization reaction can result in an article having a protein
immobilized on a three-dimensional matrix. Such matrices can have a wide
variety of uses including, but not limited to, separating and/or
immobilizing cell lines. Particularly useful proteins for these
applications include, but are not limited to, collagen, fibronectin,
gelatin, laminin, vitronectin, and/or other proteins commonly used for
cell plating.

[0070] For another example, cyclization reactions between
1,3-dipole-functional compounds and alkynes of Formula I in which X
represents a polymeric or a copolymeric group can be used, for example,
for controlling the delivery of drugs as described herein below. For
example, alkynes of Formula I in which X represents a copolymeric group
including a hydrophilic segment and a hydrophobic segment can be blended
with a polymer or a copolymer. Further, when an alkyne of Formula I in
which X represents a copolymeric group including a hydrophilic segment
and a hydrophobic segment is blended with a copolymer having a
hydrophilic segment and a hydrophobic segment, a copolymer micelle can be
formed. Particularly useful copolymers having a hydrophilic segment and a
hydrophobic segment include those of the formula
R9O--[CH2CH2O]p--[C(O)(CH2)5O]o--H,
wherein R9 represents an alkyl group (e.g., methyl), p=0 to 100
(e.g., 1 to 100), and o=0 to 100 (e.g., 1 to 100).

[0071] The copolymer micelles that include an alkyne of Formula 1 as
described herein above can advantageously be used to control the delivery
of drugs. For example, a copolymer micelle that includes an alkyne of
Formula 1 can be combined with at least one 1,3-dipole-functional drug
and allowed to react under conditions effective to form a heterocyclic
compound and attach the drug to the copolymer micelle. See, for example,
Nishiyama et al., Adv. Polym. Sci. 2006, 193:67-101; Gaucher et al., J.
Control. Release 2005, 109:169-188; Choi et al., J. Dispersion Sci. Tech.
2003, 24:475-487; Lavasanifar et al., Adv. Drug Delivery Rev. 2002,
54:169-190; and Rosier et al., Adv. Drug Delivery Rev. 2001, 53:95-108.

[0072] Further, because it does not require a toxic catalyst such as
copper, the novel cycloaddition reaction provided by the invention can be
used for labeling of living cells. For example, cells can first be
metabolically labeled with an azide-functional precursor to produce
azide-functional biomolecules (also referred to as bioconjugates) such as
azide-functional glycoproteins (also referred to as glycoconjugates). The
cells can then be contacted with an alkyne of Formula I, either in
solution or on a substrate as discussed above, under conditions to permit
labeling (via the cycloaddition reaction) of the azide-functional
biomolecules at the surface of the cell. The resulting triazole conjugate
can be detected at the cell surface, or it can be endocytosed by the cell
and detected inside the cell.

[0073] Alkynes of Formula I can also have utility for imaging applications
including, for example, as reagents for magnetic resonance imaging (MRI).
For another example, alkynes of Formula I can contain a fluorescent tag.
Alkynes of Formula I can also be useful in qualitative or quantitative
proteomics and glycomics applications utilizing mass spectrometry. The
alkyne of Formula I can be selected to contain one or more heavy mass
isotopes, such as deuterium, 13C, 15N, 35S and the like,
and then can be used to label and/or immobilize azide-functional
biomolecules as described herein.

[0074] Alkynes of Formula I can also have utility for applications such as
vaccines. For example, alkynes of Formula I can be reacted with an
azide-functional protein (e.g., an azide-functional carbohydrate, an
azide-functional peptide, and/or an azide-functional glycopeptide), and
the resulting triazole conjugate can be used as a carrier protein for the
vaccine.

[0075] The present invention is illustrated by the following examples. It
is to be understood that the particular examples, materials, amounts, and
procedures are to be interpreted broadly in accordance with the scope and
spirit of the invention as set forth herein.

EXAMPLES

Example 1

Visualizing Metabolically Labeled Glycoconjugates of Living Cells by
Copper-Free and Fast Huisgen Cycloadditions

[0077] An attractive approach for installing azides into biomolecules is
based on metabolic labeling, whereby an azide-containing biosynthetic
precursor is incorporated into biomolecules by using the cells'
biosynthetic machinery (Prescher and Bertozzi, Nat. Chem. Biol. 2005,
1:13-21). This approach has been employed for tagging proteins, glycans,
and lipids of living systems with a variety of reactive probes. These
probes can facilitate the mapping of saccharide-selective glycoproteins
and identify glycosylation sites (Hanson et al., J. Am. Chem. Soc. 2007,
129:7266-7267). Alkyne probes have also been used for cell-surface
imaging of azide-modified biomolecules, and a particularly attractive
approach involves the generation of a fluorescent probe from a
nonfluorescent precursor by a [3+2] cycloaddition (Sivakumar et al., Org.
Lett. 2004, 6:4603-4606).

[0078] The cellular toxicity of the Cu1 catalyst has precluded
applications wherein cells must remain viable (Link and Tirrel, J. Am.
Chem. Soc. 2003, 125:11164-11165), and hence there is a great need for
the development of Cu1-- free [3+2] cycloadditions (Turner et al.,
J. Am. Chem. Soc. 1973, 95:790-792; Agard et al., J. Am. Chem. Soc. 2004,
126:15046-15047; vanBerkel et al., Chem-BioChem 2007, 8:1504-1508). In
this respect, alkynes can be activated by ring strain, and, for example,
constraining an alkyne within an eight-membered ring creates 18
kcalmol-1 of strain, much of which is released in the transition
state upon [3+2] cycloaddition with an azide (Turner et al., J. Am. Chem.
Soc. 1973, 95:790-792; Agard et al., J. Am. Chem. Soc. 2004,
126:15046-15047). As a result, cyclooctynes such as 1 react with azides
at room temperature without the need for a catalyst (FIG. 1). The
strain-promoted cycloaddition has been used to label biomolecules without
observable cytotoxicity (Agard et al., J. Am. Chem. Soc. 2004,
126:15046-15047). The scope of the approach has, however, been limited
because of the slow rate of reaction (Agard et al., ACS Chem. Biol. 2006,
1:644-648). Appending electron-withdrawing groups to the octyne ring can
increase the rate of strain-promoted cycloadditions; however, currently
Staudinger ligation with phosphine 2 offers the most attractive reagent
for cell-surface labeling with azides.

[0079] It was envisaged that 4-dibenzocyclooctynols such as compound 3
would be ideal for labeling living cells with azides because the aromatic
rings are expected to impose additional ring strain and conjugate with
the alkyne, thereby increasing the reactivity of the alkyne in metal-free
[2+3] cycloadditions with azides. The compound should, however, have
excellent stability because the ortho hydrogen atoms of the aromatic
rings shield the alkyne from nucleophilic attack. Furthermore, the
hydroxy group of 3 provides a handle for the incorporation of tags such
as fluorescent probes and biotin.

[0080] Compound 3 could be prepared easily from known (Jung et al., J.
Org. Chem. 1978, 43:3698-3701; Jung and Miller, J. Am. Chem. Soc. 1981,
103:1984-1992) 3-hydroxy-1,2:5,6-dibenzocycloocta-1,5,7-triene (4) by
protection of the hydroxy group as a TBS ether to give 5, which was
brominated to provide dibromide 6 in a yield of 60% (Scheme 1; FIG. 2).
The TBS protecting group was lost during the latter transformation, but
the bromination was low yielding when performed on alcohol 4.
Dehydrobromination of 6 by treatment with LDA in THF at 0° C.
(Seitz et al., Angew. Chem. 1969, 81:427-428; Seitz et al., Angew. Chem.
Int. Ed. Engl. 1969, 8:447-448) gave the target cyclooctyne 3 in a yield
of 45%.

[0081] Compound 3 has an excellent, long shelf life and after treatment
did not react with nucleophiles such as thiols and amines. However, upon
exposure to azides a fast reaction took place and gave the corresponding
triazoles in high yield. For example, triazoles 10-13 were obtained in
quantitative yields as mixtures of regioisomers by reaction of the
corresponding azido-containing sugar and amino acid derivatives with 3 in
methanol for 30 minutes (Scheme 2; FIG. 3 and FIG. 4). The progress of
the reaction of 3 with benzyl azide in methanol and in a mixture of
water/acetonitrile (1:4 v/v) was monitored by 1H NMR spectroscopy by
integration of the benzylic proton signals, and second-order rate
constants of 0.17 and 2.3 respectively, were determined. The rate
constant of the reaction with 3 in acetonitrile/water is approximately
three orders of magnitude greater than that with cyclooctyne 1.

[0082] Having established the superior reactivity of 3, we focused our
attention on the preparation of a derivative of 4-dibenzocyclooctynol (9;
Scheme 1; FIG. 2), which is modified with biotin. Such a reagent should
make it possible to visualize biomolecules after metabolically labeling
cells with an azido-containing biosynthetic precursor, followed by
cycloaddition with 9 and treatment with avidin modified with a
fluorescence probe. Alternatively, biotinylation of glycoconjugates with
9 should make it possible to isolate these derivatives for glycocomics
studies using avidin immobilized on a solid support. Compound 9 could
easily be prepared by a two-step reaction involving treatment of 3 with
4-nitrophenyl chloroformate to give activated intermediate 7, followed by
immediate reaction with 8. 4-dibenzocyclooctynol (9) may also be
functionalized with a fluorescent tag to yield a fluorescent derivative
(Scheme 3; FIG. 5).

[0083] Next, Jurkat cells were cultured in the presence of 25 micromolar
N-azidoacetylmannosamine (Ac4ManNAz) for three days to metabolically
introduce N-azidoacetylsialic acid (SiaNAz) moieties into glycoproteins
(Luchansky and Bertozzi, Chem-BioChem 2004, 5:1706-1709). As a negative
control, Jurkat cells were employed that were grown in the absence of
Ac4ManNAz. The cells were exposed to a 30 micromolar solution of
compound 9 for various time periods, and after washing, the cells were
stained with avidin- fluorescein isothiocyanate (FITC) for 15 minutes at
4° C. The efficiency of the two-step cell-surface labeling was
determined by measuring the fluorescence intensity of the cell lysates.
For comparison, the cell-surface azido moieties were also labeled by
Staudinger ligation with biotin-modified phosphine 2 followed by
treatment with avidin-FITC. The labeling with 9 was almost complete after
an incubation time of 60 minutes (FIG. 6a).

[0084] Interestingly, under identical conditions phosphine 2 (Agard et
al., ACS Chem. Biol. 2006, 1:644-648) gave significantly lower
fluorescent intensities, indicating that cell surface labeling by
Staudinger ligation is slower and less efficient. In each case, the
control cells exhibited very low fluorescence intensities, demonstrating
that background labeling is negligible. It was found that the two-step
labeling approach with 9 had no effect on cell viability, as determined
by morphology and exclusion of trypan blue (data not shown; FIG. 7).

[0085] The concentration dependence of the cell-surface labeling was
studied by incubation of cells with various concentrations of 2 and 9
followed by staining with avidin-FTIC (FIG. 6b). As expected, cells
displaying azido moieties showed a dose-dependent increase in
fluorescence intensity. Reliable fluorescent labeling was achieved at a 3
micromolar concentration of 9; however, optimal results were obtained at
concentrations ranging from 30 to 100 micromolar. No increase in labeling
was observed at concentrations higher than 100 micromolar owing to the
limited solubility of 9.

[0086] Next, attention was focused on visualizing azido-containing
glycoconjugates of living cells by confocal microscopy. Thus, adherent
Chinese hamster ovary (CHO) cells were cultured in the presence of
Ac4ManNAz (100 micromolar) for three days. The resulting
cell-surface azido moieties were treated with 9 (30 micromolar) for 1
hour and then with avidin-AlexaFluor488 for 15 minutes at 4° C. As
expected, staining was observed only at the surface (FIG. 8), and the
labeling procedure was equally efficient when performed at either ambient
temperature or 4° C. Furthermore, blank cells exhibited very low
fluorescence staining, confirming that background labeling is negligible.

[0087] Cell-surface glycoconjugates are constantly recycled by
endocytosis, and to monitor this process, metabolically labeled cells
were reacted with 9 and avidin-AlexaFluor488 according to the standard
protocol and incubated at 37° C. for 1 hour before examination by
confocal microscopy. We observed that a significant quantity of labeled
glycoproteins had been internalized into vesicular compartments.

[0088] At the completion of these studies, Bertozzi and co-workers
reported a difluorinated cyclooctyne (DIFO) that reacts with azides at
almost the same reaction rate as compound 3 (Baskin et al., Proc. Natl.
Acad. Sci. USA 2007, 104:16793-16797). DIFO linked to AlexaFluor was
employed to investigate the dynamics of glycan trafficking. It was found
that after incubation for 1 hour, labeled glycans colocalized with
markers for endosomes and Golgi.

[0089] 4-Dibenzocyclooctynols such as 3 and 9 have several advantageous
features for researchers such as ease of chemical synthesis and the
possibility to further enhance the rate of cycloaddition by
functionalization of the aromatic moieties. Modifying the aromatic rings
may also offer an exciting opportunity to obtain reagents that become
fluorescent upon [3+2] cycloaddition with azido- containing compounds,
which will make it possible to monitor in real time the trafficking of
glycoproteins and other biomolecules in living cells.

[0090] General Methods and Materials

[0091] Chemicals were purchased from Aldrich and Fluka and used without
further purification. Dichloromethane was distilled from CaH2 and
stored over molecular sieves 4 Å. Pyridine was distilled from
P2O5 and stored over molecular sieves 4 Å. THF was
distilled form sodium. All reactions were performed under anhydrous
conditions under an atmosphere of Argon. Reactions were monitored by thin
layer chromatography (TLC) on Kieselgel 60 F254 (Merck). Detection was by
examination under ultraviolet (UV) light (254 nm) or by charring with
5° A sulfuric acid in methanol. Flash chromatography was performed
on silica gel (Merck, 70-230 mesh). Iatrobeads (60 micrometers) were
purchased from Bioscan. 1H NMR (1D, 2D) and 13C NMR were
recorded on a Varian Mere 300 spectrometer and on Varian 500 and 600 MHz
spectrometers equipped with Sun workstations. 1H and 13C NMR
spectra were recorded in CDCl3, and chemical shifts (5) are given in
ppm relative to solvent peaks (1H, δ 7.24; 13C, δ
77.0) as internal standard for protected compounds. Negative ion matrix
assisted laser desorption ionization time of flight (MALDI-TOF) were
recorded on a VOYAGER-DE Applied Biosystems using dihydrobenzoic acid as
a matrix. High-resolution mass spectra were obtained using a VOYAGER-DE
Applied Biosystems in the positive mode by using 2,5-dihydroxyl-benzoic
acid in THF as matrix.

[0097] 3-Hydroxy-7,8-didebydro-1,2:5,6-dibenzocyclooctene (2.2 mg, 0.01
mmol) was dissolved in CH3OH (1 mL) and an azide (3-azidopropyl
2,3,4,6-tetra-O-acetate-α-D-mannopyranoside,
1-O-[dimethyl(1,1,2-trimethylpropyl)silyl]-4,6-O-isopropylidene-2-azido-2-
-deoxy-β-Dglucopyranose,
4,7,8-tri-O-acetyl-5-acetamido-9-azido-2,3-anhydro-3,5,9-tri-deoxy-D-glyc-
ero-D-galacto-non-2-enonic methyl ester, and
4-azido-N-[(1,1-dimethylethoxy)carbonyl]-Lphenylalanine, 1.0 equivalents)
was added. The reaction was monitored by TLC, and after stirring the
reaction mixture for 30 minutes at room temperature, the reaction had
gone to completion. The solvents were evaporated under reduced pressure
and the residue was purified by silica gel column chromatography to
afford the desired products 10-13 respectively in quantitative yields.

[0105] Synthetic compounds 2 and 9 were reconstituted in DMF and stored at
80° C. Final concentrations of DMF never exceeded 0.56% to avoid
toxic effects.

Cell Surface Azide Labeling and Detection by Fluorescence Intensity

[0106] Human Jurkat cells (Clone E6-1; ATCC) were cultured in RPMI 1640
medium (ATCC) with L-glutamine (2 mM), adjusted to contain sodium
bicarbonate (1.5 g L-1), glucose (4.5 g L-1), HEPES (10 mM),
and sodium pyruvate (1.0 mM) and supplemented with penicillin (100 u
mL-1)/streptomycin (100 micrograms mL-1; Mediatech) and fetal
bovine serum (FBS, 10%; Hyclone). Cells were maintained in a humid 5% CO?
atmosphere at 37° C. Jurkat cells were grown in the presence of
peracetylated N-azidoacetylmannosamine (Ac4ManNaz; 25 micromolar final
concentration) for 3 days, leading to the metabolic incorporation of the
corresponding N-azidoacetyl sialic acid (SiaNAz) into their cell surface
glycoproteins. Jurkat cells bearing azides and untreated control cells
were incubated with the biotinylated compounds 2 and 9 (0-100 micromolar)
in labeling buffer (DPBS, supplemented with FBS (1%)) for 0-180 minutes
at room temperature. The cells were washed three times with labeling
buffer and then incubated with avidin conjugated with fluorescein
(Molecular Probes) for 15 minutes at 4° C. Following three washes
and cell lysis, cell lysates were analysed for fluorescence intensity
(485 ex /520 em) using a microplate reader (BMG Labtech). Data points
were collected in triplicate and are representative of three separate
experiments. Cell viability was assessed at different points in the
procedure with exclusion of trypan blue.

Cell Labeling and Detection by Fluorescence Microscopy

[0107] Chinese hamster ovary (CHO) cells (Clone K1; ATCC) were cultured in
Kaighn's modification of Ham's F-12 medium (F-12K) with L-glutamine (2
mM), adjusted to contain sodium bicarbonate (1.5 g L-1) and
supplemented with penicillin (100 u mL-1)/streptomycin (100
micrograms mL-1 and FBS (10%). Cells were maintained in a humid 5%
CO, atmosphere at 37° C. CHO cells were grown in the presence of
Ac4ManNaz (100 micromolar final concentration) for 3 days to
metabolically incorporate SiaNAz into their cell surface glycoproteins.
CHO cells bearing azides and untreated control cells were then
transferred to a glass coverslip and cultured for 36 hours in their
original medium. Live CHO cells were treated with the biotinylated
compound 9 (30 micromolar) in labeling buffer (DPBS, supplemented with
FBS (1%)) for 1 hour at 4° C. or at room temperature, followed by
incubation with avidin conjugated with Alexa Fluor 488 (Molecular Probes)
for 15 minutes at 4° C. Cells were washed 3 times with labeling
buffer and fixed with formaldehyde (3.7% in PBS) or incubated for 1 hour
at 37° C. before fixation. The nucleus was labeled with the far
red fluorescent TO-PRO-3 dye (Molecular Probes). The cells were mounted
with PermaFluor (Thermo Electron Corporation) before imaging. Initial
analysis was performed on a Zeiss Axioplan2 fluorescent microscope.
Confocal images were acquired using a 60× (NA1.42) oil objective.
Stacks of optical sections were collected in the z dimensions. The step
size, based on the calculated optimum for each objective, was between
0.25 and 0.5 micrometers. Subsequently, each stack was collapsed into a
single image (z-projection). Analysis was performed offline using ImageJ
1.39f software (National Institutes of Health, USA) and Adobe Photoshop
CS3 Extended Version 10.0 (Adobe Systems Incorporated), whereby all
images were treated equally.

[0109] An attractive approach for installing azides into biomolecules is
based on metabolic labeling whereby an azide containing biosynthetic
precursor is incorporated into biomolecules using the cells' biosynthetic
machinery (Prescher and Bertozzi, Nat. Chem. Biol. 2005, 1, 13). This
approach has been employed for tagging proteins, glycans, and lipids of
living systems with a variety of reactive probes. These probes can
facilitate the mapping of saccharide-selective glycoproteins and identify
glycosylation sites (Hanson et al., J. Am. Chem. Soc. 2007, 129, 7266).
Alkyne probes have also been used for cell surface imaging of
azide-modified bio-moleculcs and a particularly attractive approach
involves the generation of a fluorescent probe from a non-fluorescent
precursor by a [3+2] cycloaddition (Sivakumar et al., Org. Lett. 2004, 6,
4603).

[0110] We describe here reagents including an alkyne fragment, a cleavable
linker fragment, and biotin. Such compounds are expected to be valuable
for biological research. Thus, the alkyne fragment of the reagent can
react with various biomolecules containing an azide fragment to give
stable triazole adducts. The biotin fragment gives an opportunity to
retrieve the tagged compounds by affinity chromatography using
immobilized avidin. The cleavable linker allows the release of tagged and
captured biomolecules for analysis. For example, released proteins or
glycoproteins can be characterized by standard proteomics or glycomics
analysis (Too, Expert Rev. Proteomics 2007, 4, 603; Bantscheff et al.,
Anal. Bioanal. Chem. 2007, 389, 1017; Lau et al., Proteomics 2007, 7,
2787). Release of the proteins and glycoproteins is much more practical
than previously reported analysis of biomolecules attached to immobilized
avidin (Hanson et al., J. Am. Chem. Soc. 2007, 129, 7266). Compound 21 is
an example of the new class of reagent (FIG. 9). It contains a
4-dibenzocyclooctynol fragment for reaction with azides, a disulfide,
which can be cleaved with reducing reagents such as dithiothreitol (DTT),
and biotin.

[0111] The quantification of differences between physiological states of a
biological system is a technically challenging task in proteomics (Too,
Expert Rev. Proteomics 2007, 4, 603; Bantscheff et al., Anal. Bioanal.
Chem. 2007, 389, 1017; Lau et al., Proteomics 2007, 7, 2787). In
addition, to the classical methods of differential protein gel or blot
staining by dyes and fluorophores, mass-spectrometry-based quantification
methods is gaining popularity. Most of the latter methods employ
differential stable isotope labeling to create a specific mass tag that
can be recognized by a mass spectrometer and at the same time provide the
basis for quantification. These mass tags can be introduced into proteins
or peptides by (i) metabolical labeling, (ii) by chemical means, (iii)
enzymatically, or (iv) by spiking with synthetic peptide standards.

[0112] Reagents composed of an alkyne, a cleavable linker and biotin can
be employed to introduce mass tags into proteins, glycoproteins and other
biomolecules containing an azide fragment. Thus, by employing reagents
such as 21 and 22, different mass tags can be introduced to quantify
proteins, glycoproteins, glycopeptides, peptides and carbohydrates. The
chemical synthesis of 21 and 22 is depicted in Schemes 4 and 5,
respectively (FIGS. 10 and 11). Various alkyne moieties, cleavable
linkers and biotin derivatives are depicted in FIG. 12 and alkyne and
reactive diene derivatives are depicted in FIG. 13.

Example 3

Fast Click Reactions for Labeling of Living Cells and Nanoparticles

An Alterative Approach for Preparing 4-dibenzocyclooctynol and Using this
Compound for the Preparation of Amine Containing Click Reagents

[0113] 4-Dibenzocyclooctynol 45 could be prepared by an alternative
synthetic route (Scheme 6; FIG. 14). Thus, known of dibenzosuberenone
(41) was treated trimethylsilyl diazomethane in the presence of
BF3.OEt2 in CH2Cl2 (20 ml) at -10 "C to give
6H-Dibenzo[a,e]cyclooctatrien-5-one (42) in good yield. The ketone of 42
was reduced with sodium borohydride in a mixture of ethanol and THF to
give alcohol 43, which could be converted into 4-dibenzocyclooctynol 45
by bromination of the double bond followed by elimination of the
resulting compound 44 by treatment LDA in TI-IF. Compound 45 could be
oxidized to the corresponding ketone 46 by employing Dess-Martin reagent.

[0114] Compounds 45 and 46 were converted into amine containing
derivatives 49, 50 and 51. The attraction of these compounds is that the
can easily he derivatized with various probes such as fluorescent tags
and biotin. Furthermore, the amine gives an easy chemical handle for
attachment to polymeric supports. Thus, alcohol 45 was converted into
p-nitrophenyl ester 49 by reaction with 4-nitro-phenyl chloroformate (0.4
g, 2 mmol) and pyridine. The target compound compound 49 was obtained by
reaction of 49 with an excess of tris(ethylene glycol)-1,8-diamine.
Compound 50 was obtained by reaction of 46 with bromoacetic acid in the
presence of lithium diisopropylamide in tetrahydrofuran followed by
condensation of the resulting acid 48 with tris(ethylene
glycol)-1,8-diamine in DMF in the presence of the coupling reagent HATU
and the base DIPEA. Finally, derivative 51 was prepared by oxime
formation be reaction of ketone 51 with
N-{2-[2-(2-amino-ethoxy)-ethoxy]-ethyl}-2-aminooxy-acetamide (84 mg,
0.251 mmol) in the presence of acetic acid and in a mixture of methanol
and dichloromethane. A feature of 51 is that the oxime linkage can be
cleaved by treatment with aqueous acid to detach the captured compound
from the click reagent.

Reaction Kinetics of Cycloaddition of Derivatives of 4-dibenzocyclooctynol

[0125] A number of analogs (63-68) of 4-dibenzocyclooctynol (61) were
prepared and the influence of these modifications on the reaction rate of
the cycloaddition with benzyl azide was determined by integration of the
benzylic proton signals in 1H NMR spectrum. FIG. 15 shows the second
order constant of compounds 61-68. A surprising finding was that compound
62, which does not have a hydroxyl function, reacts approximately
70-times slower that the analogous 4-dibenzocyclooctynol (61). Acylation
of the hydroxyl of 61 such as in compounds 63 and 64, led to a slow
reduction in reaction rate. Alkylation of 61, as in compound 65, also
resulted in a slower rate of reaction. Compound 66, which has a
gem-difluoride reacted at a similar rate as compound 61. Interestingly,
ketone 67 reacts with a slightly higher reaction rate than 1. Oxime 68
has a similar reaction than 61. These results demonstrate that
modification of the hydroxyl of 61 can have a dramatic influence on the
rate of cycloaddition.

Modification of Macromolecules and Nano-Material Using Cycloadditions with
4-dibenzocyclooctynol

[0140] The Cu(I) catalyzed 1,3-dipolar cycloaddition of azides with
terminal alkynes to give stable triazoles has been employed for tagging a
variety of biomolecules including proteins, nucleic acids, lipids, and
saccharides. This reaction has also been used to modify polymers and
nanoscale materials. Potential difficulties to remove Cu(I), which is
highly cytotoxic, complicates the use of the 1,3-dipolar cycloaddition
for conjugation of compounds or material for biological or medical
application. The use of 4-dibenzocyclooctynol instead of a terminal
alkyne for cycloadditions with azides should overcome this problem.

[0141] To demonstrate the use of 4-dibenzocyclooctynol in bioconjugation,
co-block polymers 83 and 84 were prepared. These materials were employed
to form organomicelles in water and it was shown that
4-dibenzocyclooctyne fragment of these materials can be reacted was with
azido containing molecules (FIG. 20A, B).

[0142] It is well known that co-block polymers composed of a polyester and
polyethyleneglycol fragment self-assemble in water to form
organomicelles. These nano-materials have attracted attention as drug
delivery devises. Derivatization of organomicelles with, for example,
tissue or tumor targeting moieties may lead to smart drug delivery
devises. In addition, modification of organomicelles with fluorescent
tags or MRI reagents, such as biotin, will be valuable for imaging
purposes (FIG. 20C).

[0143] Copolymerization of polyethylene glycol methyl ether (81) or azide
(82) (MW ˜2000 Da) with caprolactone in the presence of a catalytic
amount of SnOct gave copolymers 83 and 84, respectively (Scheme 1 ; FIG.
21). The azido fragment of 84 was reduced with triphenylphosphine and the
amine of the resulting polymer 85 was reacted with 86 and 87 to give
dibenzocyclooctyl derivatives 88 and 89, respectively. A mixture of 83
and 88 or 89 (9/1, w/w) dissolved in a small amount of THF were added to
water. Cryo-TEM showed that organomicelles that have a diameter of
approximately 40A were formed. The resulting micelles were incubated with
azido-containing saccharide 90 and after a reaction time of 24 hours,
unreacted saccharide was removed by dialysis. The micelles were analyzed
for sugar content by hydrolysis with TFA followed by quantification by
high pH anion exchange chromatography. It was established that
approximately 45% of the cyclooctynes were modified by saccharides.

[0144] It is to be expected that compound 84 can also be employed for the
formation of micelles and the azido moieties of the resulting azides
employed in cycloaddition with compounds modified with a
dibenzocyclooctyl fragment.

Experimental

[0145] Synthesis of PEG44-b-PCL26 83. PEG45-b-PCL23
block copolymers were synthesized as reported. A predetermined volume
(12.0 mL) of ε-caprolactone monomer was placed in a flask
containing an amount (9.0 g) of PEG 81 under an argon atmosphere. Then, a
drop of SnOct was added. After cooling to liquid-nitrogen temperature,
the flask was evacuated for 12 hours, sealed off, and kept at 130°
C. for 24 hours. The synthesized polymers were dissolved in THF,
recovered by precipitation by cold hexane, and dried under vacuum at room
temperature. The degree of polymerization of the PCL was calculated by
1H NMR relative to the degree of polymerization of the PEG 81.

[0146] Synthesis of azide-PEG44-b-PCL26 84. Azide-PEG-b-PCL was
synthesized by a one-pot cationic ring opening polymerization at
130° C. under a stream of argon adopting a previously reported
method for the preparation of PEG-b-PCL with some modifications. Briefly,
a predetermined volume (3.3 mL) of ε-caprolactone monomer was
placed in a flask containing a preweighed amount (2.5 g) of azide-PEG-OH
82 under a nitrogen atmosphere. Then a drop of SnOct was added. After
cooling to liquid-nitrogen temperature, the flask was evacuated, sealed
off, and kept at 130° C. for 24 hours. The synthesized polymers
were then dissolved in THF, recovered by precipitation into cold hexane,
and dried under vacuum at room temperature. The number average molecular
weight (Mn) of azide-PEO44-b-PCL26 84 block copolymer was
determined by 1H NMR.

[0147] Synthesis of amine-PEG44-b-PCL26 85. Pd/C (10 wt. % on
activated carbon, 50 mg) was added to a solution of
azide-PEG44-b-PCL26 84 (200 mg)in EtOH and HOAc (50 μL),
after which H2 was bubbled through the solution for 1 hr followed by
stirring under an H2 atmosphere for 16 hours. The mixture was
filtered, concentrated in vacuum. The residues were then dissolved in
THF, recovered by precipitation into cold hexane, and dried under vacuum
at room temperature to afford amine-PEG44-b-PCL26 85.

[0150] It has been found that 4-dibenzocyclooctynol can react in the
absence of catalyst or promoter at ambient temperature with 1,3-dipoles
such as nitrones and acyl diazo derivatives, which can provide unique
opportunities for bioconjugation reactions.

[0152] Thus nitrones 91-95 were mixed with 4-dibenzocyclooctynol and after
a reaction time of 3 minutes to 3.5 hours the corresponding
2,3-dihydro-issoxazole cycloaddition products were isolated in almost a
quantitative yield. It can be seen in FIG. 18 that the chemical nature of
the nitrone has a dramatic impact of the reaction rate. In particular
electron poor nitrones 93 and 94 react at much faster rates than
corresponding azides.

Experimental

[0153] General method for calculating second order rate constants by NMR.
Substrates were dissolved separately in the appropriate solvent and mixed
1:1 at 6 mM concentrations. Percent conversion was monitored both by
disappearance of starting material and appearance of the two
regioisomeric products as determined by integration at multiple chemical
shifts. Second order rate constants for the reaction were determined by
plotting the 1/[substrates] versus time and analysis by linear
regression. Second order rate constants correspond to one half of the
determined slope.

[0154] The complete disclosure of all patents, patent applications, and
publications, and electronically available material (e.g., GenBank amino
acid and nucleotide sequence submissions; and protein data bank (pdb)
submissions) cited herein are incorporated by reference. The foregoing
detailed description and examples have been given for clarity of
understanding only. No unnecessary limitations are to be understood
therefrom. The invention is not limited to the exact details shown and
described, for variations obvious to one skilled in the art will be
included within the invention defined by the claims.

Patent applications by Geert-Jan Boons, Athens, GA US

Patent applications by Jun Guo, Athens, GA US

Patent applications by Margaretha Wolfert, Athens, GA US

Patent applications by Xinghai Ning, Athens, GA US

Patent applications by University of Georgia Research Foundation, Inc.

Patent applications in class PEPTIDES OF 3 TO 100 AMINO ACID RESIDUES

Patent applications in all subclasses PEPTIDES OF 3 TO 100 AMINO ACID RESIDUES